Proof of Concept Study for Increasing Tenascin-C-Targeted Drug Delivery to Tumors Previously Subjected to Therapy: X-Irradiation Increases Tumor Uptake

Abstract

In treatment-refractory cancers, tumor tissues damaged by therapy initiate the repair response; therefore, tumor tissues must be exposed to an additional burden before successful repair. We hypothesized that an agent recognizing a molecule that responds to anticancer treatment-induced tissue injury could deliver an additional antitumor agent including a radionuclide to damaged cancer tissues during repair. We selected the extracellular matrix glycoprotein tenascin-C (TNC) as such a molecule, and three antibodies recognizing human and murine TNC were employed to evaluate X-irradiation-induced changes in TNC uptake by subcutaneous tumors. TNC expression was assessed by immunohistochemical staining of BxPC-3 tumors treated with or without X-irradiation (30 Gy) for 7 days. Antibodies against TNC (3-6, 12-2-7, TDEAR) and a control antibody were radiolabeled with ¹¹¹In and injected into nude mice having BxPC-3 tumors 7 days after X-irradiation, and temporal uptake was monitored for an additional 4 days by biodistribution and single-photon emission computed tomography with computed tomography (SPECT/CT) studies. Intratumoral distribution was analyzed by autoradiography. The immunohistochemical signal for TNC expression was faint in nontreated tumors but increased and expanded with time until day 7 after X-irradiation. Biodistribution studies revealed increased tumor uptake of all three ¹¹¹In-labeled antibodies and the control antibody. However, a statistically significant increase in uptake was evident only for ¹¹¹In-labeled 3-6 (35% injected dose (ID)/g for 30 Gy vs. 15% ID/g for 0 Gy at day 1, p < 0.01), whereas limited changes in ¹¹¹In-labeled TDEAR2, 12-2-27, and control antibody were observed (several % ID/g for 0 and 30 Gy). Serial SPECT/CT imaging with ¹¹¹In-labeled 3-6 or control antibody provided consistent results. Autoradiography revealed noticeably stronger signals in irradiated tumors injected with ¹¹¹In-labeled 3-6 compared with each of the nonirradiated tumors and the control antibody. The signals were observed in TNC-expressing stroma. Markedly increased uptake of ¹¹¹In-labeled 3-6 in irradiated tumors supports our concept that an agent, such as an antibody, that recognizes a molecule involved in tissue injury repair, such as TNC, could enhance drug delivery to tumor tissues that have undergone therapy. The combination of antibody 3-6 coupled to a tumoricidal drug and conventional therapy has the potential to achieve better outcomes for patients with refractory cancer.

Keywords: cancer stroma; injured tissue; radioisotope; tissue remodeling; treatment resistance.

Figure 1

Schematic structure of tenascin-C (TNC) and the known binding sites of antibodies. TNC contains epidermal growth factor (EGF)-like repeats and fibronectin type III (FNIII) domains. Alternative splicing occurs between the fifth and sixth FNIII domains. The known binding sites of the three antibodies are denoted by solid lines under the domains. The antibodies 3–6 and 12–2–7 recognize the EGF-like repeats and TDEAR2 binds to the alternative splicing region.

Figure 2

Immunohistochemical staining for TNC in BxPC-3 tumors. Paraffin-embedded sections were stained with anti-TNC antibody 3–6 (n = 3 per group). Shown are representative images of tumors that had been irradiated with X-rays (30 Gy) or not irradiated.

Figure 3

Binding of ¹¹¹In-labeled anti-tenascin-C (TNC) antibody 3–6 to BxPC-3 cells (n = 3 per number of cells).

Figure 4

Schedule for the biodistribution and SPECT/CT (single-photon emission computed tomography with computed tomography) imaging studies. ¹¹¹In-labeled antibodies were intravenously injected into mice bearing BxPC-3 tumors at day 7 after X-ray exposure (0 or 30 Gy). BD = biodistribution; Img = SPECT/CT imaging.

Figure 5

Time-activity curves of ¹¹¹In-labeled antibodies in nonirradiated tumors (black circles) and tumors irradiated with 30 Gy (white circles) of X-rays. Mice (n = 5 per time point) were intravenously injected with 37 kBq (kilobecquerel) of an ¹¹¹In-labeled anti-TNC antibody (3–6, 12–2–7, and TDEAR2) or a rat nonspecific antibody as a control. Biodistribution experiments were conducted after 30 min and on days 1, 2, and 4 post-injection. Data are expressed as the mean ± S.D. (n = 5). * p < 0.05, ** p < 0.01 (nonirradiated vs. 30 Gy). The uptakes in organs and tissues are shown in Table 1, Table 2, Table 3 and Table 4.

Figure 6

Coronal SPECT/CT images of nude mice bearing BxPC-3 tumors (n = 1 per group). The imaging study was conducted after 30 min and on days 1, 2, 3, and 4 after intravenous injection with 1.85 MBq ¹¹¹In-labeled control antibody or antibody 3–6. Blue and yellow arrowheads indicate tumors. The numbers under tumors indicate the mean of % ID/g in the corresponding tumors.

Figure 7

Autoradiography of ¹¹¹In-labeled antibodies and H&E (hematoxylin and eosin) staining of BxPC-3 tumors (six sections per group). Tumors that were not irradiated or irradiated with 30 Gy of X-rays were sampled on day 1 post-injection with 1.85 MBq ¹¹¹In-labeled control antibody or antibody 3–6. (A) Representative H&E-stained sections (left panels), merged images (center panels), and autoradiographic images (ARG, right panels). Scale Bar: 2.5 mm. (B) Quantitative analysis of autoradiographic images. ** p < 0.01; N.S., not significant., a.u., arbitrary unit.

Figure 8

TNC-immunohistochemical stained sections adjacent to autographic images. Autoradiography of BxPC-3 tumor sections from mice that had been injected with ¹¹¹In-labeled anti-TNC antibody 3–6, with subsequent staining with H&E. The adjacent sections were immunostained with anti-TNC antibody 3–6. The autographic and H&E images are the same as those shown in Figure 7. Nonirradiated tumors (A) and those irradiated with 30 Gy (B) of X-rays were sampled at day 1 post-injection with 1.85 MBq of ¹¹¹In-labeled antibody 3–6.

Figure 9

Immunohistochemical staining for TNC with antibody 3–6 (left) or 12–2–7 (right) in adjacent sections. Shown are representative images (n = 6 per antibody).